Abstract Aging is an inevitable risk factor for cardiovascular disease. Profound understanding of mechanisms underlying the early stages of cardiovascular aging is essential for the development of novel therapeutics. Therefore, animal models which closely reflect the human condition are highly sought after. Here, we investigated natural cardiovascular aging in a non-human primate, comparing healthy young-adult and aged common marmosets ( Callithrix jacchus ). Despite preservation of most cardiac functional parameters in aged animals, significant histological alterations were found including fibrosis and microvascular rarefaction. Molecular phenotyping by single-nuclei RNA-sequencing revealed activation of cardiac stress, pro-inflammatory and fibrotic gene programs in aged hearts. Importantly, proteomic analysis of cardiac extracellular vesicles revealed a cardioprotective cargo in young animals while functionally demonstrating pro-angiogenic properties on human cardiac microvascular endothelial cells. Finally, large vessel atherosclerosis was strikingly evident in aged animals and elucidated by bulk RNA-sequencing. Overall, the aging marmoset offers a great potential for translational cardiovascular research.

Neutrophils play a key role in autoimmune diseases like rheumatoid arthritis, contributing to tissue damage through rapid recruitment and activation. In this study, we investigated the regulatory properties of two receptor-like tyrosine phosphatases (RPTPs), CD45 and CD148, in inflammatory arthritis. Using an in vivo mouse model of K/BxN serum transfer-induced arthritis, we found that CD45 and CD148 feature distinct regulatory properties during inflammatory arthritis. CD45 is required for neutrophil infiltration, cytokine release, and reactive oxygen species production, whereas CD148 deficiency leads to a delayed onset of arthritis but unaltered overall neutrophil infiltration and reduced ROS production. Furthermore, we could demonstrate that activation of Src family kinases in neutrophils is differentially regulated by CD45 and CD148 in a stimulus-dependent manner. Summarizing, our results suggest that CD45 is positively involved, while CD148 is positively and negatively involved in neutrophil recruitment and function during inflammatory arthritis.

In the original publication [...].

Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell transcriptome profiling of hearts with inducible cardiomyocyte-specific Wnt activation (β-cat Δex3 ) as well as with compensatory and failing hypertrophic remodeling. We show that functional enrichment analysis points to an involvement of extracellular vesicles (EVs) related processes in hearts of β-cat Δex3 mice. A proteomic analysis of in vivo cardiac derived EVs from β-cat Δex3 hearts has identified differentially enriched proteins involving 20 S proteasome constitutes, protein quality control (PQC), chaperones and associated cardiac proteins including α-Crystallin B (CRYAB) and sarcomeric components. The hypertrophic model confirms that cardiomyocytes reacted with an acute early transcriptional upregulation of exosome biogenesis processes and chaperones transcripts including CRYAB, which is ameliorated in advanced remodeling. Finally, human induced pluripotent stem cells (iPSC)-derived cardiomyocytes subjected to pharmacological Wnt activation recapitulated the increased expression of exosomal markers, CRYAB accumulation and increased PQC signaling. These findings reveal that secretion of EVs with a proteostasis signature contributes to early patho-physiological adaptation of cardiomyocytes, which may serve as a read-out of disease progression and can be used for monitoring cellular remodeling in vivo with a possible diagnostic and prognostic role in the future.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non–small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease.

Exosomes are small bi-lipid membranous vesicles (30–150 nm) containing different biological material such as proteins, lipids and nucleic acid. These small vesicles, inducing a cell to cell signaling pathway, are able to mediate multidirectional crosstalk to maintain homeostasis or modulate disease processes. With their various contents, exosomes sort and transfer specific information from their origin to a recipient cell, from a tissue or organ in the close proximity or at distance, generating an intra-inter tissue or organ communication. In the last decade exosomes have been identified in multiple organs and fluids under different pathological conditions. In particular, while the content and the abundance of exosome is now a diagnostic marker for cardiovascular diseases, their role in context-specific physiological and pathophysiological conditions in the cardiovascular system remains largely unknown. We summarize here the current knowledge on the role of exosomes as mediators of cardiovascular diseases in several pathophysiological conditions such as atherosclerosis and diabetes. In addition, we describe evidence of intercellular connection among multiple cell type (cardiac, vasculature, immune cells) as well as the challenge of their in vivo analysis.

Over the last few years, the interest in extracellular vesicles (EVs) function has exponentially grown. However, methods for isolating these small vesicles from tissue are still not trivial. Few protocols that allow EV isolation from whole tissue samples, including the heart, are available and they are based on organ perfusion unsing Langendorff method. In this work, aiming at analysing in vivo biology of small EVs, we implemented a simple method to obtain enrichment of these vesicles from murine heart tissue. We tested a titration of Liberase for tissue digestion, which was subjected to differential ultracentrifugation combined with iodixanol cushion and presented the step-by-step procedure of this protocol. Validation was done with Nanoparticle Tracking Analysis, transmission Electron Microscope and Western Blot analysis of EV markers and organelle contaminants. Furthermore, we tested the suitability of the protocol for isolating EVs from heart tissue obtained from a pre-clinical translational non-human primate animal model. Therefore, this protocol should be suitable for isolating vesicle from human heart tissue. Additionally, this method could potentially be applied beyond heart tissue.

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

Cardiovascular diseases are the leading cause of mortality worldwide. Understanding the mechanisms at the basis of these diseases is necessary in order to generate therapeutic approaches. Recently, cardiac tissue engineering and induced pluripotent stem cell (iPSC) reprogramming has led to a skyrocketing number of publications describing cardiovascular regeneration as a promising option for cardiovascular disease treatment. Generation of artificial tissue and organoids derived from induced pluripotent stem cells is in the pipeline for regenerative medicine. The present review summarizes the multiple approaches of heart regeneration with a special focus on iPSC application. In particular, we describe the strength of iPSCs as a tool to study the molecular mechanisms driving cardiovascular pathologies, as well as their potential in drug discovery. Moreover, we will describe some insights into novel discoveries of how stem-cell-secreted biomolecules, such as exosomes, could affect cardiac regeneration, and how the fine tuning of the immune system could be a revolutionary tool in the modulation of heart regeneration.

Abstract The ALK inhibitor crizotinib showed promising therapeutic efficacy for relapsed/refractory Anaplastic Large Cell Lymphoma (R/R ALCL). However, a fraction of ALK+ R/R ALCL patients do not achieve complete remission due to crizotinib resistance that develops within the first 3 months of therapy. In patients that achieve complete remission, crizotinib discontinuation causes rapid disease relapses due to the expansion of persister lymphoma cells never completely eradicated by the ALK inhibitor. There is, in fact, growing evidence that ALK+ ALCL can persist for years in patients being undetectable. ALCL grows around blood and lymphatic vessels in the lymph node. We hypothesize that this perivascular niche provides pro-survival signals contributing to ALK+ ALCL persistence and TKI resistance. By RNA-seq analysis on ALK+ ALCL cells and scRNA-seq analysis in one ALK+ ALCL primary sample, we found that ALK+ cells expressed the C-C chemokine receptor type 7 (CCR7), while endothelial cells and fibroblasts expressed the unique CCR7 ligands, the chemokine (C-C motif) ligand 19 (CCL19) and 21 (CCL21). Therefore, we explored whether the CCL19/21-CCR7 chemokine-receptor signaling axis could be involved in the persistence of ALK+ ALCL cells during ALK inhibitor treatment. We show that treatment with crizotinib caused upregulation of CCR7 in ALK+ ALCL cells via STAT3, as demonstrated by ChIP-seq data. Besides, stimulation of ALK+ ALCL cells with both CCL19/21 potently activated the MAPK signaling and sustained MAPK activation during ALK inhibition by crizotinib. Mechanistically, we demonstrate that this MAPK activation was mediated by PI3Kγ-dependent CCR7 signaling. This effect was more marked in human ALK+ ALCL cells that express high levels of PI3Kγ, while it was strongly reduced in murine lymphoma PI3KγKO cells, generated from NPM-ALK transgenic mice crossed with PI3KγKO mice. Treatment with the PI3Kγ/δ dual inhibitor duvelisib abrogated the MAPK phosphorylation induced by CCL19/21. When we knocked-out the CCR7 gene via CRISPR/Cas9, human ALK+ ALCL showed markedly reduced activation of the MAPK pathway upon stimulation with CCL19/21. Next, we developed a microphysiological model of ALCL cells in the perivascular niche with a 3D vasculature using a microfluidic chip. In this model, ALCL cells circulate inside the chip in continuous contact with a perfusable vasculature. We, then, demonstrated that the presence of endothelial cells conferred resistance to crizotinib and sustained cell viability of CCR7WT cells, whereas the protective effect was lost in CCR7KO cells. In in vivo experiments, CCR7 was required for lymphoma cell survival and diffusion to the brain during crizotinib treatment. Overall, our results suggest that the perivascular niche could promote the survival of ALK+ ALCL persister cells and protect them from the effect of ALK TKIs via the CCL19/21-CCR7 axis. The disruption of this survival axis could contribute to eradicating minimal residual disease in combination with ALK TKI. Citation Format: Cristina Mastini, Marco Campisi, Carlotta Costa, Chiara Ambrogio, Giulia Germena, Silvia Peola, Cinzia Martinengo, Enrico Patrucco, Ines Mota, Maddalena Arrigoni, Martina Olivero, Raffaele Calogero, Valeria Chiono, Roger D. Kamm, Emilio Hirsch, Jon C. Aster, Claudia Voena, Roberto Chiarle. The perivascular niche protects ALK+ lymphoma cells from ALK inhibition through the CCL19/21-CCR7 axis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT018.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the beta(2)-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of beta(2)-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-alpha deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3-dependent conformational activation of LFA-1.

During inflammation, leukocyte recruitment has to be tightly controlled to prevent overwhelming leukocyte infiltration, activation, and, consequently, organ damage. A central regulator of leukocyte recruitment is Rac1. In this study, we analyzed the effects of the RacGAP ArhGAP15 on leukocyte recruitment. Using ArhGAP15-deficient mice, reduced neutrophil adhesion and transmigration in the TNF-alpha-inflamed cremaster muscle and a prolongation of chemokine-dependent leukocyte adhesion could be observed. In a murine model of sterile kidney injury, reduced neutrophil infiltration, and serum creatinine levels were apparent. Further in vitro and in vivo analyses revealed a defective intravascular crawling capacity, resulting from increased affinity of the beta(2)-integrin Mac-1 after prolonged chemokine stimulation of neutrophils. LFA-1 activity regulation was not affected. Summarizing, ArhGAP15 specifically regulates Mac-1, but not LFA-1, and affects leukocyte recruitment by controlling postadhesion strengthening and intravascular crawling in a Mac-1-dependent manner. In conclusion, ArhGAP15 is involved in the time-dependent regulation of leukocyte postadhesion in sterile inflammation.

Mobilization of neutrophils from the bone marrow determines neutrophil blood counts and thus is medically important. Balanced neutrophil mobilization from the bone marrow depends on the retention-promoting chemokine CXCL12 and its receptor CXCR4 and the egression-promoting chemokine CXCL2 and its receptor CXCR2. Both pathways activate the small guanosine triphosphatase Rac, leaving the role of this signaling event in neutrophil retention and egression ambiguous. On the assumption that active Rac determines persistent directional cell migration, we generated a mathematical model to link chemokine-mediated Rac modulation to neutrophil egression time. Our computer simulation indicated that, in the bone marrow, where the retention signal predominated, egression time strictly depended on the time it took Rac to return to its basal activity (namely, adaptation). This prediction was validated in mice lacking the Rac inhibitor ArhGAP15. Neutrophils in these mice showed prolonged Rac adaptation and cell-autonomous retention in the bone marrow. Our model thus demonstrates that mobilization in the presence of two spatially defined opposing chemotactic cues strictly depends on inhibitors shaping the time course of signal adaptation. Furthermore, our findings might help to find new modes of intervention to treat conditions characterized by excessively low or high circulating neutrophils.

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15−/− mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.

• Gnb isoforms are centrally involved in Rac1-dependent chemokine-induced LFA-1 activation. • Plcb2 and Plcb3 function nonredundantly to produce inositol triphosphate with subsequent calcium flux leading to LFA-1 activation. Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the a and b subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstreamsignaling, relatively little is knownabout their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1,GNB2, GNB4, andGNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads toRas-relatedC3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipaseCb2 (Plcb2) and Plcb3. They act nonredundantly to produce inositol triphosphate–mediated intracellular Ca flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcb2-, Plcb3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcb2, and Plcb3 are critically involved in integrin activation and leukocyte arrest. (Blood. 2016;127(3):314-324)

Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. Src family kinases (SFKs) activation is required for integrin and chemokine signaling as well as immune cell function. The receptor-like protein tyrosine phosphatase CD45 positively regulates chemoattractant signaling acting on SFK activity. To further investigate the role of CD45 in neutrophil recruitment and function, we analyzed transgenic mice carrying a single point mutation (CD45E613R), which constitutively activates CD45. By using intravital microscopy experiments, we demonstrated that different steps of the leukocyte recruitment cascade were affected in CD45E613R mutant mice. The rolling velocity of CD45E613R mutant neutrophils was decreased compared with wild-type neutrophils that subsequently resulted in an increased number of adherent cells. The analysis of β2 integrins LFA-1 and macrophage-1 Ag (Mac-1) showed that in CD45E613R mutant neutrophils LFA-1 adhesiveness was impaired, and avidity was enhanced, whereas Mac-1 adhesiveness was increased. Because of the increased Mac-1 adhesiveness, neutrophil crawling was impaired in CD45E613R mutant compared with wild-type neutrophils. In an Escherichia coli lung infection model, CD45E613R mice displayed a decreased neutrophil recruitment into the alveolar compartment, which resulted in an increased number of CFUs in the lung. Our data demonstrate that the CD45E613R mutation modulates integrin activation and leukocyte recruitment during inflammation.

Rac and PI3Ks are intracellular signal transducers able to regulate multiple signaling pathways fundamental for cell behavior. PI3Ks are lipid kinases that produce phosphorylated lipids which, in turn, transduce extracellular cues within the cell, while Rac is a small G protein that impacts on actin organization. Compelling evidence indicates that in multiple circumstances the 2 signaling pathways appear intermingled. For instance, phosphorylated lipids produced by PI3Ks recruit and activate GEF and GAP proteins, key modulators of Rac function. Conversely, PI3Ks interact with activated Rac, leading to Rac signaling amplification. This review summarizes the molecular mechanisms underlying the cross-talk between Rac and PI3K signaling in 2 different processes, cell migration and ROS production.